Viral vector encoding pigment epithelium-derived factor

ABSTRACT

The present invention provides a viral vector comprising a nucleic acid sequence encoding pigment epithelium-derived factor (PEDF) or a therapeutic fragment thereof. The nucleic acid sequence is operably linked to regulatory sequences necessary for expression of PEDF or a therapeutic fragment thereof. Preferably, the viral vector is an adenoviral vector or an adeno-associated viral vector. Also preferably, the viral vector further comprises one or more additional nucleic acid sequences encoding therapeutic substances other than PEDF.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 09/599,997, filed Jun. 23, 2000, which claims thebenefit of U.S. Provisional Patent Application No. 60/181,743, filedFeb. 9, 2000.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a viral vector comprising a nucleicacid encoding pigment epithelium derived factor.

BACKGROUND OF THE INVENTION

Gene therapy is the next best hope for the treatment and prevention of abroad array of human diseases. Since the first gene therapy clinicaltrials started in 1990, more than 300 clinical protocols have beenapproved and an estimated $200 million is spent by the NationalInstitutes of Health (NIH) to develop the tools and techniques necessaryto practice gene therapy (Marshall, Science, 269, 1050-1055 (1995); andAnderson, Nature, 392, 25-30 (1998)). The potential of treating diseaseat its most basic level is staggering. Yet, many hurdles exist ingenetic research before the unlimited potential of gene therapy can berealized in the clinic. Obstacles associated with the wide-spreadacceptance of gene therapy include obtainment of long term geneexpression, efficient nucleic acid delivery, transduction of bothdividing and non-dividing cells, target cell specificity, safety, andconstruction of inexpensive expression vectors for use in gene therapyprotocols. Gene therapy research can be divided into two major areas:techniques and tools. Gene therapy techniques that are currently understudy include methods of gene transfer in vivo and ex vivo, cell culturemethods, methods of identifying appropriate target cells, methods ofquantifying and qualifying gene expression in vivo, and the like.

In addition to perfecting techniques employed in gene therapy, muchresearch has been focused on the tools of gene transfer for the purposeof treating or preventing disease. Many in the art have called foradditional research into the improvement of gene transfer vectors,regulatory sequences, and producer cell lines. A great deal ofenthusiasm surrounds the use of synthetic gene delivery vehicles andnaked DNA. In fact, the first expression vector used for gene transferwas naked DNA. Naked DNA, e.g., plasmids, have virtually unlimitedcapacity and are relatively simple to construct. Plasmids aregenetically engineered circular double-stranded DNA molecules that areinexpensive and easy to produce, and can transduce any type of gene orfunctional nucleic acid into cells. Yet, the level of expressionefficiency of plasmids is poor, plasmids are not easily taken up by hostcells, and plasmids are easily degraded when exposed to hightemperatures, enzymes, chemicals, mechanical stress, and the like. Thus,to increase the efficiency of gene transfer and vector stability, nakedDNA is often complexed with liposomes or other molecules. Liposomes arevesicle-type structures wherein fluid is encapsulated by a lipidbilayer. While the liposomes used for plasmid-mediated gene transferstrategies have various compositions, they are typically syntheticcationic lipids. Due to the negative charge of DNA, naked DNA isattracted to the positively-charged surface of liposomes. Naked DNA alsocan be conjugated to other molecules, such as proteins, in order tofacilitate DNA uptake. The proteins associated with DNA allow targetingof the nucleic acid molecule to a particular cell type, as well asincrease plasmid uptake. Liposome- and molecular conjugate-mediated genetransfer is a great deal more efficient than transfection ofnon-complexed, naked DNA.

Clearly, several advantages exist in using naked DNA in gene therapyprotocols. Plasmids are largely undetected by the body's innate immunesystem and, therefore, are not readily cleared by the body. In addition,plasmids are non-infectious and are rarely mutagenic. As such, naked DNAis believed by some to be the ideal mode of gene transfer for purposesof gene therapy. Yet, even when complexed with facilitators, efficiencyof host cell transfection and subsequent expression of transgenes isrelatively low. For instance, although liposome-mediated gene transfermay introduce plasmids into host cells, the majority of the transferredDNA is lost, most likely due to lysosomal degradation (French, Herz, 18,222-229 (1993)). As sufficient expression of therapeutic genes to treatdisease is a major obstacle in gene therapy, other means of genetransfer are needed in order to ensure the success of gene therapyprotocols.

In addition to the identification and development of ideal vectors,another tool needed for the success of gene therapy in the clinic istherapeutic factors and the nucleic acids that encode them. Ideally, atherapeutic factor for use in gene therapy has utility in treating anumber of afflictions, and can be delivered and expressed in vivo. Onesuch factor, pigment epithelium-derived factor (PEDF), has recently beenidentified and realized to have both neurotrophic and anti-angiogenicproperties. Regrettably, PEDF is almost solely generated in human fetusretinal cells. The poor production of PEDF from retinal pigmentepithelial (RPE) cells and the scarcity of the source tissue of PEDFcomplicates the use of this potentially valuable therapeutic factor inthe clinic.

Given the hurdles associated with gene therapy, in particular thedifficulties associated with efficient expression of appropriatetherapeutic factors, there remains a need in the art for an expressionvector comprising a coding sequence for a therapeutic factor thatpotentially can aid in the treatment of a number of afflictions. Thepresent invention provides such an expression vector. This and otheradvantages of the present invention will become apparent from thedetailed description provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a viral vector comprising a nucleic acidsequence encoding pigment epithelium-derived factor (PEDF) or atherapeutic fragment thereof. The nucleic acid sequence is operablylinked to regulatory sequences necessary for expression of PEDF or atherapeutic fragment thereof. Preferably, the viral vector is anadenoviral vector or an adeno-associated viral vector. Also preferably,the viral vector further comprises one or more additional nucleic acidsequences encoding therapeutic substances other than PEDF or atherapeutic fragment thereof.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to a viral vector comprising a nucleicacid sequence encoding pigment epithelium-derived factor (PEDF) or atherapeutic fragment thereof. The nucleic acid sequence is operablylinked to regulatory sequences necessary for expression of PEDF or atherapeutic fragment thereof. The combined efficiency of viral vectorsto deliver nucleic acids to host cells and the therapeutic potential ofPEDF had not been realized prior to the present invention. As such, thepresent invention provides a powerful tool for the prophylactic andtherapeutic treatment of disease, as well as gene therapy and diseaseresearch.

Viral vectors for use in the present invention include, for example,retroviral vectors, herpes simplex virus (HSV)-based vectors,parvovirus-based vectors, e.g., adeno-associated virus (AAV)-basedvectors, AAV-adenoviral chimeric vectors, and adenovirus-based vectors.Any of these viral vectors can be prepared using standard recombinantDNA techniques described in, e.g., Sambrook et al., Molecular Cloning, aLaboratory Manual, 2d edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N.Y. (1994).

Retrovirus is an RNA virus capable of infecting a wide variety of hostcells. Upon infection, the retroviral genome integrates into the genomeof its host cell and is replicated along with host cell DNA, therebyconstantly producing viral RNA and any nucleic acid sequenceincorporated into the retroviral genome. As such, long-term expressionof a therapeutic factor(s) is achievable when using retrovirus.Retroviruses contemplated for use in gene therapy are relativelynon-pathogenic, although pathogenic retroviruses exist. When employingpathogenic retroviruses, e.g., human immunodeficiency virus (HIV) orhuman T-cell lymphotrophic viruses (HTLV), care must be taken inaltering the viral genome to eliminate toxicity to the host. Aretroviral vector additionally can be manipulated to render the virusreplication-deficient. As such, retroviral vectors are thought to beparticularly useful for stable gene transfer in vivo. Lentiviralvectors, such as HIV-based vectors, are exemplary of retroviral vectorsused for gene delivery. Unlike other retroviruses, HIV-based vectors areknown to incorporate their passenger genes into non-dividing cells and,therefore, can be of use in treating persistent forms of disease.

HSV-based viral vectors are suitable for use as an expression vector tointroduce nucleic acids into numerous cell types. The mature HSV virionconsists of an enveloped icosahedral capsid with a viral genomeconsisting of a linear double-stranded DNA molecule that is 152 kb. Mostreplication-deficient HSV vectors contain a deletion to remove one ormore intermediate-early genes to prevent replication. Advantages of theherpes vector are its ability to enter a latent stage that can result inlong-term DNA expression, and its large viral DNA genome that canaccommodate exogenous DNA inserts of up to 25 kb. Of course, thisability is also a disadvantage in terms of short-term treatmentregimens. However, one of ordinary skill in the art has the ability todetermine the appropriate vector for a particular situation. For adescription of HSV-based vectors appropriate for use in the presentinventive methods, see, for example, U.S. Pat. Nos. 5,837,532;5,846,782; 5,849,572; and 5,804,413 and International PatentApplications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.

The viral vector of the present invention can be an AAV vector. AAVvectors are viral vectors of particular interest for use in gene therapyprotocols. AAV is a DNA virus, which is not known to cause humandisease. The AAV genome is comprised of two genes, rep and cap, flankedby inverted terminal repeats (ITRs), which contain recognition signalsfor DNA replication and packaging for the virus. AAV requiresco-infection with a helper virus (i.e., an adenovirus or a herpesvirus), or expression of helper genes, for efficient replication. AAVcan be propagated in a wide array of host cells including human, simian,and rodent cells, depending on the helper virus employed. AAV vectorsused for administration of a therapeutic nucleic acid typically haveapproximately 96% of the parental genome deleted, such that only theITRs remain. This eliminates immunologic or toxic side effects due toexpression of viral genes. In addition, delivering the AAV rep proteinenables integration of the AAV vector comprising AAV ITRs into aspecific region of genome, if desired. Host cells comprising anintergrated AAV genome show no change in cell growth or morphology (see,for example, U.S. Pat. No. 4,797,368). As such, prolonged expression oftherapeutic factors from AAV vectors can be useful in treatingpersistent and chronic diseases.

Adenovirus (Ad) is a 36 kb double-stranded DNA virus that efficientlytransfers DNA in vivo to a variety of different target cell types.Adenovirus used in vivo is preferably made replication-deficient bydeleting one or more select genes required for viral replication. Theexpendable E3 region is also frequently deleted to allow additional roomfor a larger DNA insert. The vector can be produced in high titers andcan efficiently transfer DNA to replicating and non-replicating cells.The newly transferred genetic information remains epi-chromosomal, thuseliminating the risks of random insertional mutagenesis and permanentalteration of the genotype of the target cell. However, if desired, theintegrative properties of AAV can be conferred to adenovirus byconstructing an AAV-Ad chimeric vector. For example, the AAV ITRs andnucleic acid encoding the Rep protein incorporated into an adenoviralvector enables the adenoviral vector to integrate into a mammalian cellgenome. Therefore, AAV-Ad chimeric vectors are an interesting option foruse in the present invention.

Preferably, the viral vector of the present inventive method is anadenoviral vector. In the context of the present invention, theadenoviral vector can be derived from any serotype of adenovirus.Adenoviral stocks that can be employed as a source of adenovirus can beamplified from the adenoviral serotypes 1 through 51, which arecurrently available from the American Type Culture Collection (ATCC,Rockville, Md.), or from any other serotype of adenovirus available fromany other source. For instance, an adenovirus can be of subgroup A(e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11,14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6),subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33,36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and41), or any other adenoviral serotype. Preferably, the adenovirus is asubgroup C adenovirus, e.g., serotype 2 or 5. However, non-group Cadenoviruses also can be used to prepare replication-deficientadenoviral gene transfer vectors comprising a nucleic acid sequenceencoding PEDF or a therapeutic fragment thereof. Preferred adenovirusesused in the construction of non-group C adenoviral gene transfer vectorsinclude Adl2 (group A), Ad7 (group B), Ad9 and Ad36 (group D), Ad4(group E), and Ad41 (group F). Non-group C adenoviral vectors, methodsof producing non-group C adenoviral vectors, and methods of usingnon-group C adenoviral vectors are disclosed in, for example, U.S. Pat.Nos. 5,801,030; 5,837,511; and 5,849,561 and International PatentApplications WO 97/12986 and WO 98/53087. Adenoviral vectors, methods ofproducing adenoviral vectors, and methods of using adenoviral vectorsare disclosed in, for example, U.S. Pat. Nos. 5,851,806 and 5,994,106and International Patent Applications WO 95/34671 and WO 97/27826.

The adenoviral vector is preferably deficient in at least one genefunction required for viral replication, thereby resulting in a“replication-deficient” adenoviral vector. Preferably, the adenoviralvector is deficient in at least one essential gene function of the E1region of the adenoviral genome. In addition to a deficiency in the E1region, the recombinant adenovirus also can have a mutation in the majorlate promoter (MLP). The mutation in the MLP can be in any of the MLPcontrol elements such that it alters the responsiveness of the promoter,as discussed in International Patent Application WO 00/00628. Morepreferably, the vector is deficient in at least one essential genefunction of the E1 region and at least part of the E3 region (e.g., anXba I deletion of the E3 region). With respect to the E1 region, theadenoviral vector can be deficient in at least part of the E1a regionand at least part of the E1b region. Preferably, the adenoviral vectoris “multiply deficient,” meaning that the adenoviral vector is deficientin one or more essential gene functions required for viral replicationin each of two or more regions. For example, the aforementionedE1-deficient or E1-, E3-deficient adenoviral vectors can be furtherdeficient in at least one essential gene of the E4 region. Adenoviralvectors deleted of the entire E4 region can elicit lower host immuneresponses.

Alternatively, the adenoviral vector lacks all or part of the E1 regionand all or part of the E2 region. However, adenoviral vectors lackingall or part of the E1 region, all or part of the E2 region and all orpart of the E3 region also are contemplated herein and are well-known inthe art. In one embodiment, the adenoviral vector lacks all or part ofthe E1 region, all or part of the E2 region, all or part of the E3region, and all or part of the E4 region. Suitable replication-deficientadenoviral vectors are disclosed in U.S. Pat. Nos. 5,851,806 and5,994,106 and International Patent Applications WO 95/34671 and WO97/21826. For example, suitable replication-deficient adenoviral vectorsinclude those with at least a partial deletion of the E1a region, atleast a partial deletion of the E1b region, at least a partial deletionof the E2a region, and at least a partial deletion of the E3 region.Alternatively, the replication-deficient adenoviral vector can have atleast a partial deletion of the E1 region, at least a partial deletionof the E3 region, and at least a partial deletion of the E4 region. Oneof ordinary skill in the art will appreciate that other regions of theadenoviral genome can be deleted in order to modulate the properties ofthe adenoviral vector or create additional room for nucleic acidinserts. For example, the adenoviral DNA polymerase gene can be deletedin an E1/E3 deficient vector to produce a viable vector for genetransfer.

Therefore, in a preferred embodiment, the viral vector of the presentinvention is a multiply-deficient adenoviral vector lacking all or partof the E1 region, all or part of the E3 region, all or part of the E4region, and, optionally, all or part of the E2 region. In this regard,it has been observed that an at least E4-deficient adenoviral vectorexpresses a transgene at high levels for a limited amount of time invivo and that persistence of expression of a transgene in an at leastE4-deficient adenoviral vector can be modulated through the action of atrans-acting factor, such as HSV ICPO, Ad pTP, CMV-IE2, CMV-IE86,HIV-tat, HTLV-tax, HBV-X, AAV-Rep 78, the cellular factor from the U20Sosteosarcoma cell line that functions like HSV ICPO, or the cellularfactor in PC12 cells that is induced by nerve growth factor, amongothers. In view of the above, the at least E4 deficient adenoviralvector preferably further comprises a nucleic acid sequence encoding atrans-acting factor that modulates the persistence of expression of thenucleic acid sequence encoding PEDF or a therapeutic fragment thereof.Alternatively, the viral vector of the present invention can beco-introduced into a host cell with a second expression vectorcomprising a nucleic acid sequence encoding a trans-acting factor thatmodulates the expression of the nucleic acid sequence encoding PEDF or atherapeutic fragment thereof. Preferably, the nucleic acid sequenceencoding the trans-acting factor does not encode an adenoviral E4 regiongene product. Whether expressed from the adenoviral vector or suppliedby a second expression vector, preferably, the trans-acting factor isthe Herpes simplex infected cell polypeptide 0 (HSV ICP0).

It should be appreciated that the deletion of different regions of theviral vector, e.g., the adenoviral vector, once administered to ananimal, can alter the immune response of the animal to the vector. Inparticular, deletion of different regions can reduce the inflammatoryresponse generated by the adenoviral vector. Furthermore, the adenoviralvector's coat protein can be modified so as to decrease the adenoviralvector's ability or inability to be recognized by a neutralizingantibody directed against the wild-type coat protein, as described inInternational Patent Application WO 98/40509. Such modifications areuseful for long-term treatment of persistent or chronic disease.

Similarly, the coat protein of a viral vector, preferably an adenoviralvector, can be manipulated to alter the binding specificity orrecognition of a virus for a viral receptor on a potential host cell.For adenovirus, such manipulations can include deletion of regions ofthe fiber, penton, or hexon, insertions of various native or non-nativeligands into portions of the coat protein, and the like. Manipulation ofthe coat protein can broaden the range of cells infected by a viralvector or enable targeting of a viral vector to a specific cell type.For example, in one embodiment, the viral vector comprises a chimericcoat protein (e.g., a fiber, hexon, pIX, pIIIa, or penton protein),which differs from the wild-type (i.e., native) coat protein by theintroduction of a normative amino acid sequence, preferably at or nearthe carboxyl terminus. Preferably, the normative amino acid sequence isinserted into or in place of an internal coat protein sequence. One ofordinary skill in the art will understand that the normative amino acidsequence can be inserted within the internal coat protein sequence or atthe end of the internal coat protein sequence. The resultant chimericviral coat protein is able to direct entry into cells of the viral,i.e., adenoviral, vector comprising the coat protein that is moreefficient than entry into cells of a vector that is identical except forcomprising a wild-type viral coat protein rather than the chimeric viralcoat protein. Preferably, the chimeric virus coat protein binds aendogenous binding site present on the cell surface that is notrecognized, or is poorly recognized by a vector comprising a wild-typeviral coat protein. One direct result of this increased efficiency ofentry is that the virus, preferably, the adenovirus, can bind to andenter numerous cell types which a virus comprising wild-type coatprotein typically cannot enter or can enter with only a low efficiency.

In another embodiment of the present invention, the viral vectorcomprises a chimeric virus coat protein not selective for a specifictype of eukaryotic cell. The chimeric coat protein differs from thewild-type coat protein by an insertion of a normative amino acidsequence into or in place of an internal coat protein sequence. In thisembodiment, the chimeric virus coat protein efficiently binds to abroader range of eukaryotic cells than a wild-type virus coat, such asdescribed in International Patent Application WO 97/20051.

With respect to adenovirus, specificity of binding to a given cell canalso be adjusted by use of an adenovirus comprising a short-shaftedadenoviral fiber gene, as discussed in U.S. Pat. No. 5,962,311. Use ofan adenovirus comprising a short-shafted adenoviral fiber gene reducesthe level or efficiency of adenoviral fiber binding to its cell-surfacereceptor and increases adenoviral penton base binding to itscell-surface receptor, thereby increasing the specificity of binding ofthe adenovirus to a given cell. Alternatively, use of an adenoviruscomprising a short-shafted fiber facilitates targeting of the adenovirusto a desired cell-surface receptor by the introduction of a normativeamino acid sequence either into the penton base or the fiber knob.

The ability of a viral vector to recognize a potential host cell can bemodulated without genetic manipulation of the coat protein. Forinstance, complexing an adenovirus with a bispecific molecule comprisinga penton base-binding domain and a domain that selectively binds aparticular cell surface binding site enables one of ordinary skill inthe art to target the vector to a particular cell type.

Suitable modifications to a viral vector, specifically an adenoviralvector, are described in U.S. Pat. Nos. 5,559,099; 5,731,190; 5,712,136;5,770,442; 5,846,782; 5,926,311; 5,965,541; and 6,057,155 andInternational Patent Applications WO 96/07734, WO 96/26281, WO 97/20051,WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, and WO 00/15823.Similarly, the construction of viral vectors is well understood in theart. Adenoviral vectors can be constructed and/or purified using themethods set forth, for example, in U.S. Pat. No. 5,965,358 andInternational Patent Applications WO 98/56937, WO 99/15686, and WO99/54441. Adeno-associated viral vectors can be constructed and/orpurified using the methods set forth, for example, in U.S. Pat. No.4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).

According to the invention, the nucleic acid sequence encoding PEDF or atherapeutic fragment thereof is operably linked to regulatory sequencesnecessary for expression, i.e., a promoter. A “promoter” is a DNAsequence that directs the binding of RNA polymerase and thereby promotesRNA synthesis. A nucleic acid sequence is “operably linked” to apromoter when the promoter is capable of directing transcription of thatnucleic acid sequence. A promoter can be native or non-native to thenucleic acid sequence to which it is operably linked.

Any promoter (i.e., whether isolated from nature or produced byrecombinant DNA or synthetic techniques) can be used in connection withthe present invention to provide for transcription of the nucleic acidsequence. The promoter preferably is capable of directing transcriptionin a eukaryotic (desirably mammalian) cell. The functioning of thepromoter can be altered by the presence of one or more enhancers and/orsilencers present on the vector. “Enhancers” are cis-acting elements ofDNA that stimulate or inhibit transcription of adjacent genes. Anenhancer that inhibits transcription also is termed a “silencer.”Enhancers differ from DNA-binding sites for sequence-specific DNAbinding proteins found only in the promoter (which also are termed“promoter elements”) in that enhancers can function in eitherorientation, and over distances of up to several kilobase pairs (kb),even from a position downstream of a transcribed region. Therefore,promoter regions can vary in length and sequence and can furtherencompass one or more DNA binding sites for sequence-specific DNAbinding proteins and/or an enhancer or silencer. Enhancers and/orsilencers can similarly be present on a nucleic acid sequence outside ofthe promoter per se.

Transcription of PEDF or a therapeutic fragment thereof can be directedby a viral promoter. Suitable viral promoters are known in the art andinclude, for instance, cytomegalovirus (CMV) promoters, such as the CMVimmediate-early promoter, promoters derived from human immunodeficiencyvirus (HIV), such as the HIV long terminal repeat promoter, Rous sarcomavirus (RSV) promoters, such as the RSV long terminal repeat, mousemammary tumor virus (MMTV) promoters, HSV promoters, such as the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA,78, 144-145 (1981)) or the Lap 2 promoter, promoters derived from SV40or Epstein Barr virus, an adeno-associated viral promoter, such as thep5 promoter, an adenoviral promoter, such as the Ad2 or Ad5 major latepromoter and tripartite leader, and the like.

Many of the above-identified viral promoters are constitutive promoters.Such promoters, as well as mutations thereof, are known and have beendescribed in the art (see, e.g., Boshart et al., Cell, 41, 521-530(1985)). Other suitable promoters for use in the methods of the presentinvention include the regulatory sequences of the metallothionine gene(Brinster et al., Nature, 296, 39-42 (1982)), promoter elements fromyeast or other fungi such as the Gal 4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, and thealkaline phosphatase promoter. Similarly, promoters isolated from thegenome of mammalian cells, such as the β-actin promoter, themuscle-creatine promoter, or the elongation factor 1α (EF1α promoter,can be employed.

Instead of being a constitutive promoter, the promoter can be aninducible promoter, i.e., a promoter that is up- and/or down-regulatedin response to appropriate signals. For instance, the regulatorysequences can comprise a hypoxia driven promoter. Other examples ofsuitable inducible promoter systems include, but are not limited to, theIL-8 promoter, the metallothionine inducible promoter system, thebacterial lacZYA expression system, the tetracycline expression system,and the T7 polymerase system. Further, promoters that are selectivelyactivated at different developmental stages (e.g., globin genes aredifferentially transcribed from globin-associated promoters in embryosand adults) can be employed.

For example, the promoter sequence that regulates expression of PEDF ora therapeutic fragment thereof can contain at least one heterologousregulatory sequence responsive to regulation by an exogenous agent. Theregulatory sequences are preferably responsive to exogenous agents suchas, but not limited to, drugs, hormones, or other gene products. Forexample, the regulatory sequences, e.g., promoter, preferably areresponsive to glucocorticoid receptor-hormone complexes, which, in turn,enhance the level of transcription of PEDF or a therapeutic fragmentthereof.

The regulatory sequences also can comprise a tissue-specific promoter,i.e., a promoter that is preferentially activated in a given tissue andresults in expression of a gene product in the tissue where activated. Atypically used tissue-specific promoter is a myocyte-specific promoter.A promoter exemplary of a myocyte-specific promoter is the myosinlight-chain 1A promoter. A tissue-specific promoter for use in thepresent inventive vector can be chosen by the ordinarily skilled artisanbased upon the target tissue or cell-type. For example, if the presentinventive vector is to be administered to the eye, a promoter specificto ocular tissue, such as a rhodopsin promoter, can be employed.Examples of rhodopsin promoters include, but are not limited to, a GNATcone-transducing alpha-subunit gene promoter and an interphotoreceptorretinoid binding protein promoter.

One of ordinary skill in the art will appreciate that each promoterdrives transcription, and, therefore, protein expression, differentlywith respect to time and amount of protein produced. For example, theCMV promoter is characterized as having peak activity shortly aftertransduction, i.e., about 24 hours after transduction, then quicklytapering off. On the other hand, the RSV promoter's activity increasesgradually, reaching peak activity several days after transduction, andmaintains a high level of activity for several weeks. Indeed, sustainedprotein expression driven by an RSV promoter in an adenoviral vector isobserved in all cell types studied, including, for instance, livercells, lung cells, spleen cells, diaphragm cells, skeletal muscle cells,and cardiac muscle cells. Thus, a promoter can be selected for use inthe methods of the present invention by matching its particular patternof activity with the desired pattern and level of expression of PEDF ora therapeutic fragment thereof. Alternatively, a hybrid promoter can beconstructed which combines the desirable aspects of multiple promoters.For example, a CMV-RSV hybrid promoter combining the CMV promoter'sinitial rush of activity with the RSV promoter's high maintenance levelof activity would be especially preferred for use in many embodiments ofthe present inventive method. It is also possible to select a promoterwith an expression profile that can be manipulated by the investigator.

Also preferably, the adenoviral vector comprises a nucleic acid sequenceencoding a cis-acting factor, wherein the cis-acting factor modulatesthe expression of the nucleic acid sequence encoding PEDF or atherapeutic fragment thereof. In this regard, it has been observed thatthe persistence of a transgene in an at least E4-deficient adenoviralvector can be modulated through the action of a cis-acting factor, suchas matrix attachment region (MAR) sequences (e.g., immunoglobulin heavychain μ (murine; Jenuwein et al., Nature, 385 (16), 269 (1997)), locuscontrol region (LCR) sequences, or apolipoprotein B (human; Kalos etal., Mol. Cell. Biol., 15 (1): 198-207 (1995)), among others. MARsequences have been characterized as DNA sequences that associate withthe nuclear matrix after a combination of nuclease digestion andextraction (Bode et al., Science, 255 (5041), 195-197 (1992)). MARsequences are often associated with enhancer-type regulatory regionsand, when integrated into genomic DNA, MAR sequences augmenttranscriptional activity of adjacent nucleotide sequences. It has beenpostulated that MAR sequences play a role in controlling the topologicalstate of chromatin structures, thereby facilitating the formation oftranscriptionally-active complexes. Similarly, it is believed LCRsequences function to establish and/or maintain domains permissive fortranscription. Many LCR sequences give tissue specific expression ofassociated nucleic acid sequences. Addition of MAR or LCR sequences tothe expression vector can further enhance expression of PEDF or atherapeutic fragment thereof.

The construction of an exogenous nucleic acid operably linked toregulatory sequences necessary for expression is well within the skillof the art (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed. (1989)). With respect to promoters, nucleicacid sequences, and the like, located on a viral vector according to thepresent invention, such elements can be constructed as part of acassette, either independently or coupled. In the context of the presentinvention, a “cassette” is a particular base sequence that possessesfunctions which facilitate subcloning and recovery of nucleic acidsequences (e.g., one or more restriction sites) or expression (e.g.,polyadenylation or splice sites) of particular nucleic acid sequences.With respect to the expression of nucleic acid sequences according tothe present invention, the ordinary skilled artisan is aware thatdifferent genetic signals and processing events control levels ofnucleic acids and proteins/peptides in a cell, such as, for instance,transcription, mRNA translation, and post-transcriptional processing.Transcription of DNA into RNA requires a functional promoter, asdescribed herein.

Protein expression is dependent on the level of RNA transcription thatis regulated by DNA signals, and the levels of DNA template. Similarly,translation of mRNA requires, at the very least, an AUG initiationcodon, which is usually located within 10 to 100 nucleotides of the 5′end of the message. Sequences flanking the AUG initiator codon have beenshown to influence its recognition by eukaryotic ribosomes, withconformity to a perfect Kozak consensus sequence resulting in optimaltranslation (see, e.g., Kozak, J. Mol. Biol., 196, 947-950 (1987)).Also, successful expression of an exogenous nucleic acid in a cell canrequire post-translational modification of a resultant protein. Thus,production of a protein can be affected by the efficiency with which DNA(or RNA) is transcribed into mRNA, the efficiency with which mRNA istranslated into protein, and the ability of the cell to carry outpost-translational modification. These are all factors of which theordinary skilled artisan is aware and is capable of manipulating usingstandard means to achieve the desired end result.

Along these lines, to optimize protein production, preferably thenucleic acid sequence is operatively linked to a polyadenylation site.Also, preferably all the proper transcription signals (and translationsignals, where appropriate) are correctly arranged such that the nucleicacid sequence are properly expressed in the cells into which it isintroduced. If desired, the viral vector also can comprise splice sites(i.e., splice acceptor and splice donor sites) to facilitate mRNAproduction. Moreover, if the viral vector comprises a nucleic acidsequence encoding a protein or peptide other than PEDF or a therapeuticfragment thereof, which is a processed or secreted protein or actsintracellularly, preferably the nucleic acid sequence further comprisesthe appropriate sequences for processing, secretion, intracellularlocalization, and the like.

In certain embodiments, it may be advantageous to modulate expression ofPEDF or a therapeutic fragment thereof. An especially preferred methodof modulating expression of a nucleic acid sequence comprises additionof site-specific recombination sites on the expression vector.Contacting an expression vector comprising site-specific recombinationsites with a recombinase will either up- or down-regulate transcriptionof a coding sequence, or simultaneously up-regulate transcription onecoding sequence and down-regulate transcription of another, through therecombination event. Use of site-specific recombination to modulatetranscription of a nucleic acid sequence is described, for example, U.S.Pat. Nos. 5,801,030 and 6,063,627 and International Patent ApplicationWO 97/09439.

The viral vector of the present invention comprises a nucleic acidsequence encoding PEDF or a therapeutic fragment thereof. The nucleicacid sequence encoding PEDF can be obtained from any source, e.g.,isolated from nature, synthetically generated, isolated from agenetically engineered organism, and the like. PEDF, also named earlypopulation doubling factor-1 (EPC-1), is a secreted protein havinghomology to a family of serine protease inhibitors named serpins. PEDFis made predominantly by retinal pigment epithelial cells and isdetectable in most tissues and cell types of the body. PEDF has bothneurotrophic and anti-angiogenic properties and, therefore, is useful inthe treatment and study of a broad array of diseases. Neurotrophicfactors are thought to be responsible for the maturation of developingneurons and for maintaining adult neurons. It has been postulated thatneurotrophic factors can actually reverse degradation of neuronsassociated with, for example, vision loss. Neurotrophic factors functionin both paracrine and autocrine fashions, making them ideal therapeuticagents. In this regard, PEDF has been observed to induce differentiationin retinoblastoma cells and enhance survival of neuronal populations(Chader, Cell Differ., 20, 209-216 (1987)). PEDF further has gliastaticactivity or has the ability to inhibit glial cell growth. As discussedabove, PEDF also has anti-angiogenic activity. Anti-angiogenicderivatives of PEDF include SLED proteins, discussed in WO 99/04806. Italso has been postulated that PEDF is involved with cell senescence(Pignolo et al., J. Biol. Chem., 268 (12), 8949-8957 (1998)). PEDF isfurther characterized in U.S. Pat. No. 5,840,686 and InternationalPatent Applications WO 93/24529 and WO 99/04806.

The viral vector, e.g. the adenoviral or the adeno-associated viralvector, also can comprise a nucleic acid sequence encoding a therapeuticfragment of PEDF. One of ordinary skill in the art will appreciate thatany anti-angiogenic factor or neurotrophic factor, e.g., PEDF, can bemodified or truncated and retain anti-angiogenic or neurotrophicactivity. As such, therapeutic fragments of PEDF (i.e., those fragmentshaving biological activity sufficient to, for example, inhibitangiogenesis or promote neuron survival) also are suitable forincorporation into the present inventive viral vector. Also suitable forincorporation into the viral vector are nucleic acid sequencescomprising substitutions, deletions, or additions, but which encode afunctioning PEDF peptide or a therapeutic fragment thereof. Likewise,fusion protein comprising PEDF or a therapeutic fragment thereof and forexample, a moiety that stabilizes peptide conformation, also can bepresent into the present inventive viral vector. A functioning PEDFpeptide or a therapeutic fragment thereof prevents or amelioratesneovascularization. In that PEDF also has neurotrophic activity, afunctioning PEDF peptide or a therapeutic fragment thereof desirablypromotes neuronal cell differentiation, inhibits glial cellproliferation, and/or promotes neuronal cell survival. One of ordinaryskill in the art will understand that complete prevention oramelioration of neovascularization is not required in order to realize atherapeutic effect. Likewise, complete induction of neuron survival ordifferentiation is not required in order to realize a benefit.Therefore, both partial and complete prevention and amelioration ofangiogenesis or promotion of neuron survival is appropriate. Theordinarily skilled artisan has the ability to determine whether amodified PEDF or a fragment of PEDF has neurotrophic and anti-angiogenictherapeutic activity using, for example, neuronal cell differentiationand survival assays (see, for example, U.S. Pat. No. 5,840,686), themouse ear model of neovascularization, the rat hindlimb ischemia model,or the methods of Examples 1 and 2.

In addition to the above, the viral vector comprising a nucleic acidsequence encoding PEDF or a therapeutic fragment thereof can furthercomprise one or more additional nucleic acid sequences encoding atherapeutic substance(s) other than PEDF or a therapeutic fragmentthereof. Desirably, the expression of the therapeutic substance isbeneficial, e.g., prophylactically or therapeutically beneficial, to thehost cell. If the therapeutic substance confers a prophylactic ortherapeutic benefit to the cell, the therapeutic substance can exert itseffect at the level of RNA or protein. For example, the therapeuticsubstance can be an additional anti-angiogenic factor or neurotrophicfactor other than PEDF or a therapeutic fragment thereof, or can be apeptide other than an anti-angiogenic factor or neurotrophic factor thatcan be employed in the treatment of a disorder. Alternatively, thetherapeutic substance can be an antisense molecule, a ribozyme, aprotein that affects splicing or 3′ processing (e.g., polyadenylation),or a protein that affects the level of expression of another gene withinthe cell (i.e., where gene expression is broadly considered to includeall steps from initiation of transcription through production of aprocess protein), such as by mediating an altered rate of mRNAaccumulation or transport or an alteration in post-transcriptionalregulation. Preferably, the therapeutic substance is a neurotrophicfactor, such as ciliary neurotrophic factor (CNTF). CNTF belongs to theneuropoietic cytokines subclass of neurotrophic factors. CNTF promotesthe survival of ciliary ganglionic neurons and supports certain neuronsthat are nerve growth factor (NGF)-responsive.

Alternatively, one or more additional nucleic acid sequences encoding atherapeutic substance(s) can encode a factor associated with celldifferentiation. Preferably, the therapeutic substance is anatonal-associated peptide such as Math1 or Hath1 or a biologicallyactive fragment of either of the foregoing. Math1 is a member of themouse basic helix-loop-helix family of transcription factors and ishomologous to the Drosophila gene atonal. Hath1 is the human counterpartof Math1. Math1 has been shown to be essential for hair development andcan stimulate hair regeneration in the ear. Combining the neurotrophicproperties of PEDF and the hair cell differentiation properties of anatonal-associated peptide provides a powerful tool for the treatment andresearch of, for example, sensory disorders. Math1 is furthercharacterized in, for example, Bermingham et al., Science, 284,1837-1841 (1999) and Zheng et al., Nature Neurosci., 3 (2), 580-586(2000).

One or more additional nucleic acid sequences encoding therapeuticsubstances can encode an anti-angiogenic substance other than PEDF or atherapeutic fragment thereof. An anti-angiogenic substance is anybiological factor that prevents or ameliorates neovascularization. Oneof ordinary skill in the art will understand that the anti-angiogenicsubstance can effect partial or complete prevention and amelioration ofangiogenesis to achieve a therapeutic effect. An anti-angiogenicsubstance includes, for instance, an anti-angiogenic factor, ananti-sense molecule specific for an angiogenic factor, a ribozyme, areceptor for an angiogenic factor, and an antibody that binds a receptorfor an angiogenic factor.

Anti-angiogenic factors include, for example, angiostatin,vasculostatin, endostatin, platelet factor 4, heparinase, interferons(e.g., INF□), and the like. One of ordinary skill in the art willappreciate that any anti-angiogenic factor can be modified or truncatedand retain anti-angiogenic activity. As such, active fragments ofanti-angiogenic factors (i.e., those fragments having biologicalactivity sufficient to inhibit angiogenesis) are also useful forincorporation into the present inventive viral vector.

An anti-sense molecule specific for an angiogenic factor shouldgenerally be substantially identical to at least a portion, preferablyat least about 20 continuous nucleotides, of the nucleic acid encodingthe angiogenic factor to be inhibited, but need not be identical. Theanti-sense nucleic acid molecule can be designed such that theinhibitory effect applies to other proteins within a family of genesexhibiting homology or substantial homology to the nucleic acid. Theintroduced anti-sense nucleic acid molecule also need not be full-lengthrelative to either the primary transcription product or fully processedmRNA. Generally, higher homology can be used to compensate for the useof a shorter sequence. Furthermore, the anti-sense molecule need nothave the same intron or exon pattern, and homology of non-codingsegments will be equally effective. Antisense phosphorothiotacoligodeoxynucleotides (PS-ODNs) is exemplary of an anti-sense moleculespecific for an angiogenic factor.

Ribozymes can be designed that specifically pair with virtually anytarget RNA and cleave the phosphodiester backbone at a specificlocation, thereby functionally inactivating the target RNA. In carryingout this cleavage, the ribozyme is not itself altered and is, thus,capable of recycling and cleaving other molecules, making it a trueenzyme. The inclusion of ribozyme sequences within anti-sense RNAsconfers RNA-cleaving activity upon them, thereby increasing the activityof the constructs. The design and use of target RNA-specific ribozymesis described in Haseloffet al., Nature, 334, 585-591 (1988). Preferably,the ribozyme comprises at least about 20 continuous nucleotidescomplementary to the target sequence on each side of the active site ofthe ribozyme.

Receptors specific for angiogenic factors inhibit neovascularization bysequestering growth factors away from functional receptors capable ofpromoting a cellular response. For example, soluble VEGF-R1 (flt-1),VEGF-R2 (flk/kdr), and VEGF-R3 (flt-4) receptors, as well asVEGF-receptor chimeric proteins, compete with VEGF receptors on vascularendothelial cells to inhibit endothelial cell growth (Aiello, PNAS, 92,10457 (1995)). Preferably, the viral vector of the present inventioncomprises at least one nucleic acid sequence encoding soluble fltreceptor in addition to the nucleic acid sequence encoding PEDF or atherapeutic fragment thereof. Receptors specific for angiogenic factors,in particular the soluble fit receptor, and use thereof to inhibitangiogenesis is further described in Kendall et al., Proc. Nat. Acad.Sci. USA, 90 (22), 10705-10709 (1993), Kong et al., Hum. Gene Ther., 9,823-833 (1988), and International Patent Application WO 94/21679. Alsocontemplated are growth factor-specific antibodies and fragments thereof(e.g., Fab, F(ab′)2, and Fv) that neutralize angiogenic factors (e.g.,VEGF) or bind receptors for angiogenic factors.

The nucleic acid sequence encoding PEDF or a therapeutic fragmentthereof and any nucleic acid sequence encoding a therapeutic substanceother than PEDF or a therapeutic fragment thereof can be operably linkedto different promoters. As discussed herein, different promoters havedissimilar levels and patterns of activity. One of ordinary skill in theart will appreciate the freedom to dictate the expression of differentcoding sequences through the use of multiple promoters. Preferably, thenucleic acid sequence encoding PEDF or a therapeutic fragment thereof isoperably linked to a RSV promoter and one or more additional nucleicacids sequences is operably linked to a CMV promoter, or vice versa.Alternatively, the multiple coding sequences can be operably linked tothe same promoter to form a polycistronic element. The polycistronicelement is transcribed into a single mRNA molecule when transduced intothe ocular cell. Translation of the mRNA molecule is initiated at eachcoding sequence, thereby producing the multiple, separate peptidessimultaneously. On the other hand, the nucleic acid sequence encodingPEDF or a therapeutic fragment thereof and at least one additionalnucleic acid sequence encoding a different therapeutic substance can belinked to a bi-directional promoter.

In addition to a nucleic acid sequence encoding PEDF or a therapeuticfragment thereof and optionally a nucleic acid encoding a differenttherapeutic substance, the viral vector also can comprise a selectiongene or a nucleic acid sequence encoding marker protein, such as greenfluorescent protein or luciferase. Selection genes are useful in vectorconstruction protocols. Marker proteins also are useful in vectorconstruction and in determining vector migration. Marker proteins canfurther be used to determine points of injection or treated tissues inorder to efficiently space injections of the expression vector toprovide a widespread area of gene transfer, if desired. The additionalnucleic acid sequence(s) encoding the therapeutic substance or markerprotein can be part of an expression cassette.

It should be appreciated that any of the nucleic acid sequencesdescribed herein can be altered from their native form to increase theirtherapeutic effect. For example, a cytoplasmic form of a therapeuticnucleic acid can be converted to a secreted form by incorporating anendoplasmic reticular localization signal peptide into the encoded geneproduct. The therapeutic substance and/or PEDF or a therapeutic fragmentthereof can be designed to be taken up by neighboring cells by fusion ofthe peptide with VP22, HIV tat, and the like. This allows a cellcomprising the nucleic acid sequence to have a therapeutic effect on anumber of surrounding cells.

To facilitate storage and, optionally, administration, the viral vectordesirably is part of a pharmaceutical composition, which comprises apharmaceutically acceptable carrier and the viral vector. Any suitablepharmaceutically acceptable carrier can be used within the context ofthe present invention, and such carriers are well known in the art. Thechoice of carrier will be determined, in part, by the particular site towhich the composition is to be administered and the particular methodused to administer the composition. The composition also can compriseagents which facilitate uptake of the viral vector into host cells.Suitable composition formulations include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain anti-oxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood or intraocular fluid of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, immediately prior to use.Extemporaneous solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.Preferably, the pharmaceutically acceptable carrier is a buffered salinesolution. More preferably, the viral vector is present in apharmaceutical composition formulated to protect the expression vectorfrom damage prior to administration. For example, the pharmaceuticalcomposition can be formulated to reduce loss of the viral vector ondevices used to prepare, store, or administer the viral vector, such asglassware, syringes, or needles. The pharmaceutical composition can beformulated to decrease the light sensitivity and/or temperaturesensitivity of the expression vector. To this end, the pharmaceuticalcomposition preferably comprises a pharmaceutically acceptable liquidcarrier, such as, for example, those described above, and a stabilizingagent selected from the group consisting of polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such apharmaceutical composition will extend the shelf life of the vector andfacilitate administration of the viral vector.

In addition, one of ordinary skill in the art will appreciate that theviral vector of the present invention can be present in a compositionwith other therapeutic or biologically-active agents. For example,therapeutic factors useful in the treatment of a particular indicationcan be present. For instance, if treating vision loss, hyaluronidase canbe added to a composition to effect the break down of blood and bloodproteins in the vitreous of the eye. Factors that control inflammation,such as ibruprofen, can be part of the composition to reduce swellingand inflammation associated with in vivo administration of the viralvector. Anti-angiogenic factors, such as soluble growth factorreceptors, growth factor antagonists, i.e., angiotensin, and the likecan also be part of the composition, as well as additional neurotrophicfactors. Similarly, vitamins and minerals, anti-oxidants, andmicronutrients can be co-administered. Antibiotics, i.e., microbicidesand fungicides, can be present to reduce the risk of infectionassociated with gene transfer procedures and other disorders.

EXAMPLES

The following examples further illustrate the present invention but, ofcourse, should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a method of determining whether a PEDF proteinor fragment thereof has therapeutic activity.

An adenoviral vector deficient in one or more essential gene functionsof the E1, E3, and E4 regions of the adenoviral genome and comprising aPEDF gene or fragment thereof (Ad.PEDF) is preferably constructed as setforth in WO 99/15686 (McVey et al.).

Several in vivo models of ocular neovascularization are available.Neovascularization of the retina is obtained in, for example, neonatalanimals, i.e., neonatal mice, by exposing the mice to hypoxic conditionsshortly after birth. Several days later, the neonatal mice are exposedto standard atmospheric conditions, resulting in ischemia-inducedneovascularization of the retina.

Ad.PEDF is administered to the right eye of at least 12 day old miceanesthetized with, for example, ketamine or a combination of ketamineand xylazine via intravitreal injection. Injections are performed byforming an entrance site in the posterior portion of the eye andadministering approximately 0.1-5.0 μl of composition comprisingAd.PEDF. In most instances, an injection of the expression vector willbe administered to only one eye, while the remaining eye serves as acontrol. The mice are sacrificed at various time points afteradministration to determine the extent and duration of PEDF expressionin the retina. The right and left eyes of each animal are enucleated andeither fixed for histological analysis or prepared for PEDF expressionanalysis. If desired, detection of PEDF DNA, PEDF RNA, or PEDF proteincan be accomplished using methods well known in the art, such as PCR andblotting techniques (see, for example, Sambrook et al., supra).

To determine the effect of PEDF on neovascularization in vivo in, forexample, a human, indirect ophthalmoscopy of the retina is ideal.Stereophotographs are useful in detecting extensive neovascularization,but not appropriate for detecting subtle lesions. Other methods ofmonitoring neovascularization include fluorescein angiography, which isuseful in determining vascular leakage, color fundus photography,scanning electron microscopy of the retinal layer, and vascular casts.Any of the above techniques is appropriate for determining whether aPEDF peptide or fragment thereof has therapeutic activity. For example,a PEDF peptide or therapeutic fragment thereof desirably inhibits orameliorates neovascularization of the retina compared to animals treatedwith null-vectors or other negative controls.

Example 2

The following example demonstrates a method of determining the effect ofPEDF or a therapeutic fragment thereof on neovascularization.

An adenoviral vector deficient in one or more essential gene functionsof the E1, E3, and E4 regions of the adenoviral genome and comprising aPEDF gene (Ad.PEDF) is constructed as set forth in WO 99/15686 (McVey etal.).

An in vivo model of choroidal neovascularization can be obtained bydetaching the retina of an eye of, for example, a mouse or rabbit, anddebriding the pigmented epithelia. Choriocapillary regeneration ismonitored in both treated and untreated eyes. Ad.PEDF is administeredprior to perturbing the retinal pigment epithelial (RPE) to determinethe effect of the present inventive method in preventing choroidalneovascularization. Of course, Ad.PEDF is administered after perturbingthe retina and RPE for determining the therapeutic effect of theprocedure on neovascularization.

Choroidal neovascularization can be monitored in vivo using fundusphotography, fluorescein angiography, and/or indocyanine-greenangiography, as commonly used in the art. Using these methods, one ofordinary skill in the art is able to detect growth of new blood vesselsand vascular leakage associated with neovascularization. For researchpurposes, neovascularization also can be determined by enucleating theeyes and preparing vascular casts or examining ocular tissue viascanning electron microscopy.

All references cited herein are hereby incorporated by reference to thesame extent as if each reference was individually and specificallyindicated to be incorporated by reference and was set forth in itsentirety herein.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

1. An adeno-associated viral vector comprising a nucleic acid sequenceencoding pigment epithelium-derived factor (PEDF) or a therapeuticfragment thereof, wherein the nucleic acid sequence is operably linkedto regulatory sequences necessary for expression of PEDF or atherapeutic fragment thereof.
 2. The viral vector of claim 1, whereinthe adeno-associated viral vector comprises a genome that is deficientin the rep and cap genes.
 3. The viral vector of claim 1, wherein theadeno-associated viral vector requires co-infection with a helper virusfor replication.
 4. The viral vector of claim 3, wherein the helpervirus is an adenovirus or a herpes virus.
 5. The viral vector of claim1, wherein the regulatory sequences comprise a promoter selected fromthe group consisting of a CMV promoter, an RSV promoter, anadeno-associated virus p5 promoter, a Lap2 promoter, an EF1α promoter,and a β-actin promoter.
 6. The viral vector of claim 5, wherein theregulatory sequences comprise an RSV promoter.
 7. The viral vector ofclaim 1, wherein the regulatory sequences comprise an induciblepromoter.
 8. The viral vector of claim 1, further comprising one or moreadditional nucleic acid sequences encoding therapeutic substances otherthan PEDF or a therapeutic fragment thereof.
 9. The viral vector ofclaim 8, wherein one or more additional nucleic acid sequences encodesciliary neurotrophic factor (CNTF).
 10. The viral vector of claim 8,wherein one or more additional nucleic acid sequences encodes anatonal-associated peptide.
 11. The viral vector of claim 8, wherein oneor more additional nucleic acid sequences encodes an anti-angiogenicsubstance.
 12. The viral vector of claim 11, wherein the anti-angiogenicsubstance is a soluble receptor specific for an angiogenic factor. 13.The viral vector of claim 12, wherein the soluble receptor specific foran angiogenic factor is a soluble VEGF-R1 receptor.
 14. The viral vectorof claim 8, wherein the therapeutic substances other than PEDF or atherapeutic fragment thereof are linked to an endoplasmic reticulumlocalization signal peptide.